Thrombopoietin (TPO) is a 332 amino acid hormone produced primarily by the liver that acts by binding to the product of the c-mpl proto-oncogene. Numerous studies have established that TPO is the primary regulator of megakaryocyte (MK) and platelet production, and is critical for both steady state and emergent platelet production. We have previously examined the role of the hormone in the survival, self-renewal and expansion of hematopoietic stem cells (HSCs), performed studies that probe the structure-function relationships of TPO and the Mpl receptor, and mapped several of the signal transduction pathways employed by Mpl in primary hematopoietic cells. Based on these results we propose a scientific plan composed of 4 specific aims. In the first we will explore the molecular mechanisms responsible for stromal cell production of TPO and its relative importance to thrombopoiesis, generating a murine model in which the stroma is either the only organ to produce the hormone, or the only one not to do so. In the second set of experiments we will solve the tertiary structure of the signaling domain of Mpl, in both monomeric and dimeric conformations, providing the first look at the structural basis for how cytokine receptors initiate signaling. We will also map the sites at which TPO physically interacts with Mpl using a deuterium exchange protection strategy. In the third specific aim we will continue to study the signaling mechanisms employed by TPO/Mpl, focusing on PKC and p38 MAPK, and their role in HSCs and platelet formation. Finally, the fourth aim will explore the molecular mechanisms of HSC self-renewal and expansion promoted by TPO, testing whether genes known to affect HSC proliferation and survival, p27, hox, notch, and p53, are responsible for the TPO effects. We will use a limiting dilution transplantation assay of stem cells transduced with p27, p53, hoxB4 or notch into tpo+/+ and tpo-/- recipients, to test if these genes genetically interact with tpo, and then test whether the hormone directly affects expression of the genes, or if TPO affects their signal transduction pathways. It is hoped that the successful completion of these studies will further our understanding of hematopoiesis.